Serotonin-2 receptor-mediated regulation of release of acetylcholine by minaprine in cholinergic nerve terminal of hippocampus of rat

5-Hydroxytryptamine (5-HT) inhibited the K+-induced release of [3H]acetylcholine [( 3H]ACh) from slices of the hippocampus of the rat, dose-dependently. Minaprine (3-(2-morpholinoethylamino)-4-methyl-6-phenylpyridazine, Fig. 1) had no effect on the release of [3H]ACh. However, it inhibited the (formula; see text) Fig. 1. Chemical structure of minaprine dihydrochloride. attenuation of the release of [3H]ACh by 5-HT dose-dependently. The 5-HT2 receptor antagonists, mianserine, methysergide and spiperone, prevented the inhibitory effect of the 5-HT, as well as did minaprine. The attenuating effect of 5-HT was not mimicked by the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and was not prevented by a 5-HT1A and 5-HT1B mixed receptor antagonist, propranolol, or by the 5-HT3 receptor antagonists, cocaine and metoclopramide. Minaprine inhibited the bindings of [3H]5-HT, [3H]8-OH-DPAT and [3H]ketanserin in the hippocampus. The inhibitory effect of minaprine on the binding of [3H]ketanserin was more marked than on the binding of [3H]5-HT and [3H]8-OH-DPAT, and was non-competitive. The Ki value of minaprine for the binding of [3H]ketanserin was 2.9 microM. The inhibitory effect of 5-HT on the release of [3H]ACh was observed in the presence of tetrodotoxin. By electrolytic lesioning of the medial septum, the K+-induced release of [3H]ACh from the slices of hippocampus was significantly reduced and the release was no longer inhibited by 5-HT. The lesioning significantly decreased the binding of [3H]ketanserin in the hippocampus, with hardly any reduction in the binding of [3H]5-HT and [3H]8-OH-DPAT.(ABSTRACT TRUNCATED AT 250 WORDS)     

5-HT1B receptors modulate release of [3H]dopamine from rat striatal synaptosomes: further evidence using 5-HT moduline, polyclonal 5-HT1B receptor antibodies and 5-HT1B receptor knock-out mice

In previous paper based on classical pharmacological tools, we identified a Gi protein-coupled presynaptic 5-hydroxytryptamine (5-HT) 1B receptor causing inhibition of dopamine (DA) release in rat striatal synaptosomes. It was the aim of the present study to further explore this receptor, using 5-HT moduline, a polyclonal antibody directed against 5-HT1B receptors and 5-HT1B receptor knock-out mice. Preincubation of rat striatal synaptosomes with 5-HT moduline (0.1, 1, or 10 microM) significantly reduced the inhibitory effect of CP93,129, a selective rat 5-HT1B receptor agonist, on K+-evoked overflow of [3H]DA in a non-competitive manner: 5-HT moduline did not modify the IC50 of CP93,129, but concentration-dependently reduced the maximal inhibitory effect. Preincubation of rat striatal synaptosomes with a specific polyclonal 5-HT1B receptor antibody also resulted in a significant attenuation of the inhibitory effect of CP93,129 on K+-evoked overflow of [3H]DA. In female 129/Sv wild-type mice, CP93,129 and 5-carboxyamidotryptamine maleate (5-CT), a non-selective 5-HT1B receptor agonist, inhibited the K+-evoked [3H]DA overflow in a concentration-dependent manner. Sumatriptan, a selective rat 5-HT1D receptor agonist, did not modify the overflow of [3H]DA. SB224289, a selective 5-HT1B receptor antagonist, abolished the inhibitory effects of CP93,129 and 5-CT. The inhibitory effects of CP93,129 and 5-CT were absent in synaptosomes from 5-HT1B receptor knockout mice. No compensatory inhibition effect in mutant mice was observed using sumatriptan. In conclusion, the results show that a non-competitive antagonist of the 5-HT1B receptor concentration-dependently decreases the maximal inhibitory effect of a 5-HT1B receptor agonist on the synaptosomal K+-evoked release of [3H]DA in striatum. Moreover, a specific antibody raised against the receptor and particularly directed against a region of the receptor protein involved in signal transduction, namely the coupling with the G-protein, also antagonizes the inhibitory effect of the stimulation of 5-HT1B receptor on the release of [3H]DA. Ultimately the disruption of 5-HT1B receptor gene in 5-HT1B knock-out mice leads to a total suppression of the effect of 5-HT1B receptor agonists on [3H]DA release. These observations further support our previous observations using selective agonists/antagonists, indicating that 5-HT1B receptors control the release of neuronal DA as presynaptic heteroreceptors.     

Differential sensitivity of 5-HT1B auto and heteroreceptors

The effect of the native and rodent-selective 5-HT1B receptor agonists (5-hydroxytryptamine (5-HT) and CP93,129) on the K+-evoked overflows of [3H]5-HT, [3H]dopamine (DA) and [3H]acetylcholine (ACh) was studied in synaptosome preparations obtained from rat brain striatum or hippocampus loaded with radiolabeled neurotransmitter. The aim of the study was to compare the different potencies of the specific 5-HT1B receptor agonists to stimulate the auto and heteroreceptors and to modulate the different neurotransmitter release. Results show that under the same experimental conditions, 5-HT and CP93,129 exhibited significantly higher potencies in inhibiting the K+-evoked overflow of [3H]5-HT from synaptosomes of rat striatum (IC50=2.0+/-1.8 nM and 20.5+/-3.1 nM, respectively) than in inhibiting the K+-evoked overflow of [3H]DA from synaptosomes of the same cerebral region (IC50= 0.8+/-0.2 microM and 1.8+/-0.4 microM, respectively), or [3H]ACh from synaptosomes of hippocampus (IC50=1.7+/-0.8 microM for CP93,129). The inhibitory effects of the 5-HT1B receptor agonists on [3H] K+-overflows were antagonized by the selective 5-HT1B receptor antagonist (SB224289), further indicating that the observed effects were 5-HT1B receptor specific. Sumatriptan, a selective r5-HT1D receptor agonist, did not show any significant effect on the K+-overflow of [3H]5-HT in the range of concentrations (10(-10) to 10(-6) M), and did not affect the K+ overflow of [3H]DA or [3H]ACh at concentrations (10(-9) to 10(-4) M), which exclude the involvement of 5-HT1D receptors. These inhibitory effects of the 5-HT1B receptor agonists were highly attenuated by pertussis toxin in the three systems studied, suggesting the involvement of Gi/Go-proteins in the transduction mechanism pathway of the receptor generated signal. In conclusion, these results suggest that 5-HT1B heteroreceptors located on dopaminergic and cholinergic terminals exhibit a lower sensitivity to 5-HT1B receptor agonist and antagonist than do 5-HT1B autoreceptors. The observed difference in functional sensitivities of 5-HT1B auto- and heteroreceptors may represent important consequences in the physiological control of the release of serotonin versus that of other neurotransmitters.     

Dual effects of 5-hydroxytryptamine on the release of gamma-aminobutyric acid from myenteric neurones of the guinea-pig ileum.

The effects of 5-hydroxytryptamine (5-HT) on the release of gamma-aminobutyric acid (GABA) were examined in the longitudinal muscle-myenteric plexus (LM-MP) preparation of guinea-pig ileum. 5-HT increased the spontaneous release and inhibited the electrically-evoked release of [3H]-GABA. The 5-HT-evoked release was Ca2+-dependent and tetrodotoxin-sensitive, and was antagonized by (3 alpha-tropanyl)-1H-indole-3-carboxylic acid ester (ICS 205-930), but not by methysergide and ketanserin. The inhibitory effect of 5-HT was antagonized by methysergide, but not by ketanserin and ICS 205-930. 8-Hydroxy-2-(di-n-propylamino)tetralin mimicked the inhibitory effect of 5-HT. Thus, 5-HT may exert an excitatory effect on the enteric GABAergic neurone via the 5-HT3 receptor and an inhibitory effect via the 5-HT1A receptor.     

Two types of receptors for 5-hydroxytryptamine on the cholinergic nerves of the guinea-pig myenteric plexus.

The effects of 5-hydroxytryptamine (5-HT) on spontaneous and electrically-evoked release of [3H]-acetylcholine (ACh) from guinea-pig myenteric plexus preparations preincubated with [3H]-choline have been investigated in the absence of cholinesterase inhibitors. 5-HT caused a transient increase in spontaneous release and an inhibition of the electrically-evoked release of [3H]-ACh. The 5-HT-induced contractions of the longitudinal muscle were clearly related to the increase in spontaneous release. The inhibitory effect was not due to activation of alpha-adrenoceptors since it was also observed in the presence of tolazoline and on strips from reserpine-pretreated guinea-pigs. After desensitization of the excitatory 5-HT receptors with 5-HT or metoclopramide the effects of 5-HT on spontaneous [3H]-ACh release were largely reduced. A variety of established antagonists at neuronal 5-HT receptors (i.e. metitepine 0.1-1 microM; methysergide 1 microM; ketanserin 0.1-1 microM; MDL 72222 0.1 microM; tropacocaine 1 microM) failed to block the excitation. The inhibition by 5-HT of the electrically evoked [3H]-ACh release was competitively antagonized by metitepine (pA2 7.6) and methysergide (pA2 7.0) but not by ketanserin. Tachyphylaxis to the inhibitory action of 5-HT did not occur. The results suggest that the excitatory 5-HT receptor ('M'-receptor) differs in its pharmacological properties from other neuronal 5-HT receptors. The presynaptically located inhibitory receptor may roughly correspond to the 5-HT1 receptor subtype but probably differs from the 5-HT autoreceptor.     

Protection with phencyclidine against inactivation of 5-HT2 receptors by sulfhydryl-modifying reagents

We investigated whether phencyclidine (PCP)-induced head-twitch was antagonized in rats by ritanserin, a selective serotonin2 (5-HT2) receptor antagonist, to confirm the involvement of 5-HT neurons in PCP action and to discover whether PCP could protect the binding sites of [3H]PCP and [3H]ketanserin from the inhibitory effect of protein-modifying reagents which affect sulfhydryl groups. PCP (7.5, 10 and 12.5 mg/kg, i.p.)-induced head-twitch was completely antagonized by ritanserin (1 mg/kg, s.c.). Scatchard plots of specific [3H]PCP and [3H]ketanserin binding showed that sulfhydryl-modifying reagent, N-ethylmaleimide (NEM, 100 microM) caused a significant decrease in Bmax without changing Kd. PCP (10 microM) and ritanserin (1 microM) protected [3H]PCP and [3H]ketanserin binding sites from the decrease in the number induced by NEM (100 microM). 5-HT protected [3H]5-HT binding sites from inactivation by NEM, but PCP and ritanserin did not show any effect. On the basis of the present findings, it is concluded that PCP can interact with 5-HT2 receptors directly or allosterically, and 5-HT2 receptors may locate at PCP binding sites in membranes.     

Role of 5-HT3 receptors in basal and K(+)-evoked dopamine release from rat olfactory tubercle and striatal slices.

1. The present study was aimed at examining the role of 5-HT3 receptors in basal and depolarization-evoked dopamine release from rat olfactory tubercle and striatal slices. [3H]-dopamine ([3H]-DA) release was measured in both brain regions and endogenous dopamine release from striatal slices was also studied. 2. The selective 5-HT3 receptor agonist 2-methyl-5-HT (0.5-10 microM) produced a concentration-dependent increase in [3H]-DA efflux evoked by K+ (20 mM) from slices of rat olfactory tubercle. 1-Phenylbiguanide (PBG) and 5-HT also increased K(+)-evoked [3H]-DA efflux. 3. 5-HT (1-100 microM) increased in a concentration-dependent manner basal [3H]-DA release from olfactory tubercle and striatal slices as well as endogenous DA release from striatal slices. The selective 5-HT3 receptor agonists 2-methyl-5-HT and 1-phenylbiguanide were weaker releasing agents. In all cases, the release was Ca2+ independent and tetrodotoxin insensitive. 4. 5-HT3 receptor antagonists such as ondansetron, granisetron and tropisetron (0.2 microM) significantly blocked the enhanced K(+)-evoked [3H]-DA efflux from rat olfactory tubercle slices induced by 2-methyl-5HT. A ten fold higher concentration of the 5-HT2 receptor antagonist ketanserin was ineffective. 5. Much higher concentrations, up to 50 microM, of the same 5-HT3 receptor antagonists did not block the increase in basal [3H]-DA release from striatal or olfactory tubercle slices induced by 5-HT or the release of endogenous DA induced by 5-HT from striatal slices.2+ off     

Regulation of 5-HT2 receptors in rat cortex. Studies with a putative selective agonist and an antagonist

The phenylisopropylamine derivative 1-(2,5-dimethoxy-4-iodo-phenyl)-2-aminopropane (DOI) has been suggested recently as a selective serotonin2 (5-HT2) receptor agonist. Because of the potential importance of such a tool for investigations of 5-HT2 receptor regulation, receptor binding studies were performed in rats after acute and chronic treatment with DOI, the selective 5-HT2 antagonist ketanserin, or vehicle. Single injections of 5 or 10 mg/kg DOI reduced the Bmax of cortical sites labeled with [3H]1-(2,5-dimethoxy-4-bromo-phenyl)-2-aminopropane and [3H]ketanserin (9-32 or 32-46%, respectively). Chronic daily treatment with DOI (3-9 mg/kg) further down-regulated 5-HT2 sites in cortex identified with either [3H]ketanserin (-60%) or with [3H]DOB (-75%), without altering Kd values or affecting 5-HT1 sites. In vitro addition to the [3H]ketanserin or [3H]DOB binding assay of 10 nM to 1 microM DOI resulted in competitive inhibition, suggesting that down-regulation found in vivo was not secondary to residual drug. Chronic treatment with ketanserin (10 mg/kg) also down-regulated both [3H]ketanserin (-38%) and [3H]DOB (-58%) sites in cortex without charges in 5-HT1 sites. In naive cortex, competition experiments revealed a Ki (nM) for ( +/- )-DOI of 1.7 +/- 0.02 at sites labeled by [3H]DOB, and a KH and KL of 4.8 +/- 1.5 and 53 +/- 2 nM at sites labeled by [3H]ketanserin. These data indicate that in chronic treatment, DOI, like ketanserin, is highly selective for 5-HT2 vs 5-HT1 sites at behaviorally useful doses. However, a representative putative 5-HT2 selective agonist and antagonist have similar effects on 5-HT2 receptors labeled by agonist or antagonist radioligands.     

Inhibitory presynaptic 5-hydroxytryptamine (5-HT) receptors on the sympathetic nerves of the human saphenous vein

Superfused strips of the human saphenous vein preincubated with 3H-noradrenaline were used to investigate the influences of serotonin (5-HT) receptor agonists and antagonists on the electrically evoked tritium overflow. 5-HT and the preferential 5-HT1A receptor agonist 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin] concentration-dependently inhibited the evoked 3H overflow. The evoked 3H overflow was not affected by 0.1 or 1 mumol/l TVX Q 7821 (2-(4-[4-(2-pyrimidinyl)-1-piperazinyl]-butyl)-1,2-benzoisothiazol -3(2H)one-1,1-dioxide), which selectively binds to 5-HT1A sites; TVX Q 7821 10 mumol/l produced an increase in overflow. The inhibitory effect of 5-HT on the impulse-evoked 3H overflow was abolished by the nonselective 5-HT receptor antagonist metitepin, but was not attenuated by propranolol. Metitepin also abolished the inhibitory effect of 8-OH-DPAT on evoked 3H overflow, whereas the 5-HT2 receptor antagonist ketanserin was inactive in this respect. There was also no antagonism of the effect of 8-OH-DPAT by the alpha 2-adrenoceptor antagonist rauwolscine or the dopamine receptor antagonist flupenthixol. These results suggest that both 5-HT and 8-OH-DPAT inhibit noradrenaline release by activating inhibitory 5-HT receptors on the sympathetic nerves of the human saphenous vein. These receptors possess similarities to 5-HT1 recognition sites, but a further subclassification is not yet possible on the basis of the available data.     

Characterization of presynaptic 5-HT receptors on adrenergic nerves supplying the bovine ovarian follicle.

1. The effects of 5-hydroxytryptamine (5-HT) on contraction and release of [3H]-noradrenaline were investigated in vitro in bovine ovarian follicle strips. Using available selective agonists and antagonists, an effort was made to characterize the type of receptor mediating the inhibitory effect of 5-HT on neurogenic contraction and release of [3H]-noradrenaline by electrical field stimulation. 2. 5-Hydroxytryptamine inhibited the neurogenic contraction and release of [3H]-noradrenaline evoked by electrical field stimulation in a concentration-dependent manner. Like 5-HT, 5-carboxamidotryptamine (5-CT) and methysergide reduced the transmitter release as well as the neurogenic contraction, whereas 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) failed to inhibit both responses in concentrations up to 0.1 microM. 3. The 5-HT (1 microM)-induced inhibition of contractile responses was more evident during stimulation at low frequencies (4 and 8 Hz) than during high frequency electrical stimulation (16 and 32 Hz). 4. Methiothepin (1 microM) and methysergide (10 microM) significantly antagonized the inhibitory effect of 5-HT on the electrically evoked release of tritium, whereas cyanopindolol, MDL 72222 and ketanserin (all 0.1 microM) were without effect. In addition, ketanserin, MDL 72222, cimetidine, pyrilamine, atropine, propranolol and indomethacin were without effect on the 5-HT-induced inhibition of the neurogenic contraction. 5. It is suggested that 5-HT inhibits the electrically evoked transmitter release from adrenergic nerves in the bovine ovarian follicle wall via prejunctional 5-HT1-like receptors. This was based on the findings that 5-CT was a potent agonist, methiothepin an antagonist and the lack of effect of MDL 72222, cyanopindolol and ketanserin.     

Identity of inhibitory presynaptic 5-hydroxytryptamine (5-HT) autoreceptors in the rat brain cortex with 5-HT1B binding sites

In rat brain cortex slices preincubated with [3H]5-HT, the potencies of 17 5-HT receptor agonists to inhibit the electrically evoked 3H overflow and the affinities of 13 antagonists (including several beta-adrenoceptor blocking agents) to antagonize competitively the inhibitory effect of unlabelled 5-HT on evoked 3H overflow were determined. The affinities of the compounds for 5-HT1B and 5-HT2 binding sites in rat brain cortex membranes (labelled by [125I]cyanopindolol = [125I]-CYP in the presence of 30 mumol/l isoprenaline and [3H]ketanserin, respectively), for 5-HT1A binding sites in pig and rat brain cortex membranes (labelled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin = [3H]8-OH-DPAT) and for 5-HT1C binding sites in pig choroid plexus membranes (labelled by [3H]mesulergine) were also determined. The affinities of the drugs for the various 5-HT recognition sites ranged over 4-5 log units (the functional experiments revealed the same range of differences between the drugs). There were no significant correlations between the affinities of the drugs at 5-HT1C and 5-HT2 binding sites and their potencies or affinities, determined for the 5-HT autoreceptors. In contrast, significant correlations were found between the potencies or affinities of the drugs for the autoreceptors and their affinities at 5-HT1A or 5-HT1B binding sites; the best correlations were obtained with the 5-HT1B binding site. Some of the drugs investigated were not included in the correlation since their agonistic or antagonistic effects on the autoreceptors were weak and pEC30 or apparent pA2 values could not be determined (less than 5.5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Selectivity of serotonergic drugs for multiple brain serotonin receptors. Role of [3H]-4-bromo-2,5-dimethoxyphenylisopropylamine ([3H]DOB), a 5-HT2 agonist radioligand

The affinities of putative serotonin receptor agonists and antagonists for 5-HT1A, 5-HT1B, 5-HT1C, and 5-HT2 receptors were assayed using radioligand binding assays. The 5-HT1 sites were labeled with the agonist radioligands [3H]-8-hydroxy-2-(di-n-propylamino)-tetralin [3H]-8-OH-DPAT, [3H]-5-HT, and [3H]mesulergine. The 5-HT2 receptor was labeled with the antagonist radioligand [3H]ketanserin or the agonist radioligand [3H]-4-bromo-2,5-dimethoxyphenylisopropylamine ([3H]DOB). The apparent 5-HT1 receptor selectivity of agonist compounds was found to be 50- to 100-fold higher when the 5-HT2 receptor affinity was determined using the antagonist radioligand [3H]ketanserin than when the agonist radioligand [3H]DOB was used. Quipazine, a putative specific 5-HT2 agonist, appeared to be only 3-fold more potent at 5-HT2 than at 5-HT1A receptors when [3H]ketanserin was used as the 5-HT2 radioligand. When [3H]DOB was used as the 5-HT2 radioligand, quipazine was determined to be 100-fold more potent at 5-HT2 receptors than at 5-HT1A receptors. 1-(3-trifluoromethylphenyl)piperazine (TFMPP), a putative specific 5-HT1B receptor agonist was apparently 10-fold more potent at 5-HT1B receptors than at 5-HT2 receptors when [3H]ketanserin was used as the 5-HT2 radioligand. When [3H]DOB was used as the 5-HT2 radioligand, TFMPP was found to be equipotent at 5-HT1B and 5-HT2 receptors. Using the 5-HT2 antagonist radioligand [3H]ketanserin, a similar pattern of underestimating 5-HT2 receptor selectivity and/or overestimating 5-HT1A or 5-HT1B receptor selectivity was observed for a series of serotonin receptor agonists. Antagonist receptor selectivity was not affected significantly by the nature of the 5-HT2 receptor assay used. These data indicate that, by using an antagonist radioligand to label 5-HT2 receptors and agonist radioligands to label 5-HT1 receptors, the 5-HT1 receptor selectivity may be overestimated. This may be an especially severe problem in serotonin drug development as drugs that interact potently with 5-HT2 receptors have been reported to be psychoactive and/or hallucinogenic.     

Assessment of affinity and dissociation ability of a newly synthesized 5-HT2 antagonist, AT-1015: comparison with other 5-HT2 antagonists

This study investigated the binding affinities of a newly synthesized 5-HT2 antagonist, AT-1015 (N-[2-[4-(5H-dibenzo[a,d]cyclohepten-5-ylidene)-piperidino]ethyl]-1-formyl-4-piperidinecarboxamide monohydrochloride monohydrate) for [3H]ketanserin bindings to 5-HT2 receptors in the rabbit cerebral cortex membranes using the radioligand binding assay method. The affinity of this compound was also compared with other 5-HT2-selective antagonists such as ketanserin, sarpogrelate, cyproheptadine and ritanserin, and the results showed that AT-1015 has a high pKi value for the 5-HT2 receptor. The rank order of these antagonists are: ritanserin > ketanserin approximately equal to AT-1015 > cyproheptadine approximately equal to sarpogrelate. We also evaluated the dissociation ability (slow or rapid) of AT-1015 in the rabbit cerebral cortex membrane and compared it with other 5-HT2 antagonists using the radioligand binding assay method. The blockade of [3H]ketanserin binding sites in the rabbit cerebral cortex induced by ketanserin and sarpogrelate was readily reversed by washing, whereas the inhibition by AT-1015, cyproheptadine and ritanserin was not readily reversed by washing. The % of control after washing are 76.10% and 49.55% for AT-1015 at 10(-7.5) and 10(-7.0) M, 67.32% and 50.17% for cyproheptadine at 10(7.5) and 10(-7.0) M, and 72.38% and 39.80% for ritanserin at 10(-9.5) and 10(-9.0) M concentrations, respectively. Thus, these findings suggest that AT-1015 has antagonistic properties towards the 5-HT2 receptor and also shows that AT-1015 slowly dissociates from the 5-HT2 receptor, whereas, ketanserin and sarpogrelate dissociate rapidly from the 5-HT2 receptor, which do not correlate with their affinity.     

Autoradiographic analysis of 5-HT2A binding sites in the brain of Sardinian alcohol-preferring and nonpreferring rats.

The density of 5-HT2A binding sites in the brain of Sardinian alcohol-preferring (sP) and nonpreferring (sNP) rats was evaluated, using [3H]ketanserin for quantitative autoradiography. The highest [3H]ketanserin binding levels were found in the anterior olfactory nucleus, prefrontal cortex, medial prefrontal cortex, post-genual anterior cingulate cortex, insular cortex and claustrum. Statistically significant differences between sP and sNP rats were found in prefrontal cortex, medial prefrontal cortex and post-genual anterior cingulate cortex, where sP rats showed about 20% lower [3H]ketanserin binding levels. No significant difference was found in other areas, although some of them showed slightly lower [3H]ketanserin binding density in sP rats. The 5-HT2A receptor agonist, (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane hydrochloride (DOI), microinjected into the medial prefrontal cortex, induced a lower number of wet dog shakes in sP than in sNP rats. These results indicate a different density of 5-HT2A binding sites, and a different functional regulation of 5-HT2A receptor mechanisms in discrete brain areas of sP, in comparison to sNP rats. These findings, and those showing lower levels of 5-HT in the frontal cortex of sP rats, suggest that altered 5-HT function in fronto-cortical areas could be linked to the genetic predisposition to high voluntary ethanol intake in these rats.     

Pharmacological characterization of the 5-HT1A, 5-HT2 and 5-HT3 receptors in the bovine ciliary muscle

We aimed to investigate the effects of serotonin (5-hydroxytryptamine, 5-HT) on the bovine ciliary muscle and subsequently to characterize and identify the subtypes of 5-HT receptors involved in the serotonin-evoked contractility muscle. The binding of [3H]ketanserin, [3H]granisetron and [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) was analyzed. All labelled compounds bound with high affinity to a single site in the membrane preparations studied. The affinity (K(d)) of the binding site was 7.5+/-1.2 nM for [3H]ketanserin, 6.9+/-0.8 nM for [3H]granisetron and 4.4+/-0.31 nM for [3H]8-OH-DPAT. The density of receptors (B(max)) was 1062+/-43.0 fmol/mg protein for [3H]ketanserin, 566+/-2.32 fmol/mg protein for [3H]granisetron and 205+/-4.63 fmol/mg protein for [3H]8-OH-DPAT. The serotonin-induced contraction appeared to be competitively antagonized by ketanserin (0.1, 1 and 10 microM) and ondansetron (0.1, 10 and 100 microM) which produced a pA(2) value of 8.5+/-0.12 and 8.0+/-0.19, respectively. 8-OH-DPAT and 5-carboxamidotryptamine (5-CT) proved to be completely ineffective. We conclude that serotonin induces bovine ciliary muscle contraction via 5-HT(2) and 5-HT(3) receptors while the 5-HT(1A) receptors, although present, do not mediate the contractile response.     

Ketanserin binds to the monoamine transporter of chromaffin granules and of synaptic vesicles

[3H]Ketanserin binding studies were performed on purified chromaffin granule membranes. Binding was found to occur on one class of sites and was temperature dependent. At 30 degrees the equilibrium dissociation constant KD was 45 nM. At 0 degrees, a KD value of 6 nM and a half-life of dissociation of 40 sec were measured. Methysergide, an antagonist of 5-hydroxytryptamine2 (5-HT2) receptors structurally unrelated to ketanserin, did not displace ketanserin binding. Tetrabenazine, an inhibitor of the monoamine transporter of chromaffin granules, displaced [3H]ketanserin binding. Conversely, ketanserin inhibited the binding of [3H] dihydrotetrabenazine, a ligand that specifically binds to the monoamine transporter. The inhibition was of the competitive type, indicating that both drugs bind to the same site. Ketanserin binding did not depend upon ATP-induced energization of chromaffin granules. ATP-dependent 5-HT uptake by chromaffin granule ghosts was inhibited by ketanserin with an IC50 value of 70 nM. A series of ketanserin derivatives were tested for their ability to displace [3H]dihydrotetrabenazine; EC50 values differed by more than 2 orders of magnitude and were not correlated to affinities on 5-HT2 receptors. In mouse brain, [3H]ketanserin was found to bind to methysergide-sensitive and to tetrabenazine-sensitive sites. In the striatum, tetrabenazine-sensitive sites represented a larger fraction than the methysergide-sensitive ones, whereas the reverse was true in the frontal cortex. It is concluded that nonspecific displaceable binding sites of [3H]ketanserin previously described in the striatum are tetrabenazine binding sites associated with the synaptic vesicle monoamine transporter.     

Cholinergic terminals in rat hippocampus possess 5-HT1B receptors mediating inhibition of acetylcholine release

The effects of 5-hydroxytryptamine (5-HT) on the release of [3H]acetylcholine ([3H]ACh) from rat hippocampal nerve endings were investigated using synaptosomes labelled with [3H]choline and depolarized in superfusion with 15 mM KCl. The release of [3H]ACh was concentration dependently inhibited by exogenous 5-HT. The concentration-response curve of 5-HT was shifted to the right in a parallel way by methiothepin. The 5-HT2 antagonists ketanserin or methysergide did not antagonize the effect of 5-HT. The 5-HT1 agonist 5-methoxy-3-[1,2,3,6-tetrahydropyridin-4-yl]-1H-indole (RU 24969) mimicked 5-HT, whereas the 5-HT1A selective agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was ineffective. When used as a 5-HT1A/5-HT1B antagonist, (-)propranolol antagonized 5-HT whereas spiperone (a 5-HT1A displacer) did not. The 5-HT1C selective antagonist mesulergine was also ineffective towards 5-HT. It can be concluded that hippocampal cholinergic terminals are endowed with inhibitory 5-HT receptors which appear to belong to the 5-HT1B subtype.     

Binding of [3H]haloperidol to dopamine D2 receptors in the rat striatum.

The present study was designed to examine the properties of [3H]haloperidol binding to dopamine D2-receptors in rat striatum membranes, displacement potencies of various chemicals and differences between the affinities of various chemicals and two new 5-hydroxytryptamine (5-HT2) receptor antagonists, MCI-9042, (+/-)-2-(dimethylamino)-1-[[o-(m-methoxyphenetyl)phenoxy]methyl]et hyl hydrogen succinate hydrochloride and one of its metabolites. The plots of specific binding for the striatum membranes obtained from the Scatchard analysis using [3H]haloperidol were monophasic when non-specific binding was determined with 10 microM chlorpromazine, and the Kd and Bmax values were 7.42 +/- 1.03 nM and 1.58 +/- 0.20 pmol (mg protein)-1 (n = 10), respectively. The displacement potencies of D2 receptor, 5-HT2 receptor, histamine H1-receptor, and adrenoceptor antagonists were characterized by [3H]haloperidol binding to D2 receptors. The pKi values of a new antiplatelet agent, MCI-9042, and its metabolite were 5.02 and 5.53, respectively, and these values were lower than those of the D2-receptor antagonists, fluphenazine, spiperone, haloperidol, prochlorperazine, chlorpromazine, thioridazine, and sulpiride. They were also lower than the pKi values of the 5-HT2-receptor antagonists, pirenperone, ketanserin, methysergide, and mianserin. We conclude that the binding site of [3H]haloperidol in the rat striatum is the D2 receptor, that MCI-9042 and its metabolite have lower affinities for D2 receptors than for 5-HT2 receptors, and that this radioreceptor assay is useful for assessing the affinities of various agents.     

5-Hydroxytryptamine and the metabolism of arachidonic acid by the lipoxygenase and cyclooxygenase of washed human platelets

During secondary aggregation, platelets release 5-hydroxytryptamine (5-HT) from their dense granule stores concurrent with arachidonic acid (AA) metabolism. To examine the hypothesis that released 5-HT has a modulatory effect on the metabolism of AA by platelets, we incubated nonaggregating washed human platelets with 5-HT in the presence of [3H]AA. Stimulation with 10(-4) M 5-HT, followed by incubation with 3 microM AA and 1 microCi [3H]AA for 5 min, resulted in a decrease in the formation of thromboxane B2 (TxB2) and 12-hydroxyheptadecatrienoic acid (HHT, P less than 0.05). The same treatment conditions and stimulation with 10(-7) to 10(-4) M 5-HT resulted in an elevation of 12-hydroxyeicosatetraenoic acid (12-HETE) formation (P less than 0.05). Treatment with the monoamine uptake inhibitor imipramine (20 microM) further increased the stimulation of 12-HETE formation observed in the presence of 10(-4) M 5-HT, suggesting that 5-HT may act at the platelet surface. A 5-HT1A receptor agonist, 8-hydroxy-dipropylaminotetralin (DPAT, 10(-6) to 10(-4) M) stimulated the formation of platelet cyclooxygenase (CO) products, whereas (+/-)1-(2,5-dimethoxy-4-iodo phenyl)-amino propane hydrochloride (DOI, 10(-6) to 10(-4) M), a 5-HT2 receptor agonist, had no significant effect on CO product formation. In addition, the 5-HT2 receptor antagonist ketanserin (10(-7) M) did not block the changes in CO or lipoxygenase metabolism induced by 5-HT. Since both DOI and DPAT stimulated 12-HETE formation whereas ketanserin was unable to reverse the 5-HT-enhanced 12-HETE formation, it seems unlikely that the stimulation of a 5-HT2 receptor is responsible for this action of 5-HT on platelets. We conclude that 5-HT depresses CO product formation while increasing 12-HETE formation through interaction with a platelet serotonergic binding site other than the 5-HT2 receptor.     

3H]-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and [3H] ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor

The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to [3H]ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding the serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both [3H]DOB and [3H]ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate [Gpp(NH)p] to this system caused a rightward shift and steepening of agonist competition curves for [3H] ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity [3H]DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that [3H]DOB and [3H]ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein. These observations are consistent with the classical view of interconvertible agonist affinity states of GTP-binding protein-coupled receptors and strongly support the "two state" over the "two receptor" model for DOB binding to the 5-HT2 receptor.